Fibrinolysis is the process of degradation of fibrin in the blood. Fibrinolysis is involved in a number of physiopathological processes and is triggered in situations when tissue plasminogen activator and plasminogen bind to fibrin, forming a ternary fibrin-plasminogen complex within which the t-PA has a high affinity for plasminogen, entraining the generation of plasmin, an enzyme which degrades fibrin into D-dimers. In the absence of fibrin, t-PA has little affinity for plasminogen, explaining the fact that circulating fibrin is not degraded.
Degradation of fibrin, or fibrinolysis, leads to the formation of degradation products especially comprising “D-dimer” fragments. Said D-dimers are associated with the E fragment from degradation of another fibrin monomer molecule forming the DDE complex, but even in that form, they are routinely termed D-dimers.
The fibrin undergoing the fibrinolysis process is formed by conversion of fibrinogen under the action of a coagulation enzyme, namely thrombin. During coagulation activation, the thrombin generated thus induces the formation of deposits of fibrin which will constitute the thrombus and the formation of soluble fibrin. To accomplish this, thrombin attacks four peptide bonds of the fibrinogen located respectively on the 2 A alpha and the 2 B beta chains, causing the liberation of two A fibrinopeptides from the two A alpha chains and the liberation of two B fibrinopeptides from the B beta chains, resulting in the formation of fibrin monomers which polymerize spontaneously into the form of a polymer by dint of hydrogen bonds established by interaction between A and B polymerization sites unmasked during liberation of the A and B fibrinopeptides and the a and b sites which are available at the ends of the gamma and beta chains respectively. The fibrin polymer is then immediately stabilized by factor XIII(a). Thrombin generation is much greater during in vitro tests than that which takes place in vivo. For this reason, the generation of fibrin monomers is much slower in the in vivo coagulation activation process than in that generated in vitro, which causes part of the monomers formed to polymerize to produce insoluble fibrin constituting the thrombus and another part of said monomers to react with fibrinogen in which the a and b sites are accessible, or with fibrinogen degradation products to produce soluble fibrin in which fibrin monomers are associated with fibrinogen.
Determining the concentration of soluble fibrin is important in order to witness the activation of coagulation in a patient. Said determination may be carried out using samples of blood or plasma obtained from a blood sample taken from a patient.
It has been shown that assaying soluble fibrin is a useful complement to assaying fibrinolysis degradation products, since soluble fibrin can detect coagulation activation which is under way while the concentration of D-dimers indicates degradation of a thrombus, even if the activation coagulation process is stopped.
In summary, the D-dimer plasma level is increased while the fibrin clot degrades in vivo. Hence, if the thrombus is present and undergoing degradation, the level of D-dimers is high, whether coagulation persists or is stopped. In contrast, the level of soluble fibrin is raised only if coagulation persists.
Compared with the level of D-dimers, specific measurement of the soluble fibrin plasma level thus allows a determination of the coagulation occurring in a patient at the moment the sample to be analyzed is taken, along with an evaluation of the coagulolytic balance.
Determining the level of D-dimers in the sample, termed the base level, is thus a reflection of the degradation of the thrombus which occurs in vivo, while determining the level of D-dimers obtained after exogenic addition of a specific fibrin thrombolytic agent represents the sum of the base D-dimers and the D-dimers deriving from degradation of soluble fibrin, also termed circulating fibrin.
International patent application WO-A-02/18628 describes a method for assaying soluble fibrin in a blood sample, necessitating bringing plasma into contact with a plasminogen activator with a high affinity for soluble fibrin (PA-Fb sp), followed by determining the level of fibrin degradation products (D-dimers). The difference between the concentration of D-dimers in a sample treated with PA-Fb sp and that of the base D-dimers determined on plasma not treated with PA-Fb sp thus represents the D-dimers linked to soluble fibrin degradation. The inventors have now observed that the method proposed in prior art International application WO-A-02/18628 may advantageously be supplemented to be carried out in the context of the diagnosis of thromboembolic venous diseases, as well as the diagnosis and monitoring of disseminated intra-vascular coagulations (DIVC). It also allows a determination of whether anti-coagulant treatment will be effective to be made.
Thromboembolic venous diseases principally comprise venous thromboses of the limbs and pulmonary embolism, the latter resulting from a complication of the first thromboses. Venous thromboses other than those of the limbs are also encountered, since all venous territories can undergo a thrombosis. The renal veins and mesenteric veins can be cited in particular among those which are at the origin of pathologies. Thromboembolic diseases such as deep venous thrombosis (DVT) and/or pulmonary embolism (PE) are life-threatening diseases and represent a large proportion of the disabilities and deaths in industrialized countries, and establishing a diagnosis of these diseases is vital in completing investigations by imaging examinations such as ultrasound imaging for the diagnosis of venous thromboses and scintography or angiography to diagnose pulmonary embolisms. These exploratory methods are tricky to carry out and cannot always be carried out rapidly enough.
As a result, there is a continuing need for defining a test allowing rapid diagnosis of thromboembolic disease in a patient, that diagnosis including the possibility of excluding that disease without necessarily having recourse to additional investigations.